Evaluation of serum‐free differentiation conditions for C2C12 myoblast cells assessed as to active tension generation capability

We have compared several serum‐free media for the differentiation of C2C12 myoblasts and assessed the extent of differentiation in several ways including as to active tension generation capability. C2C12 cells were allowed to differentiate in Dulbecco's modified Eagle's medium (DMEM) containing Ham's F‐12 (F‐12), AIM‐V (AIM), 0.2% Ultroser‐G in DMEM (Ult‐G), and 0.1% Sericin in DMEM (Sericin), compared with in DMEM supplemented with 2% horse serum (HS) or 2% calf serum (CS). C2C12 differentiation was assessed as the extent of myotube formation, glucose metabolism, protein expression, sarcomere formation, and active tension generation. All serum‐free media examined were capable of inducing myotube formation and the expression of muscle‐specific proteins. All serum‐free media except for F‐12 gave the sarcomere structure. Active tension generation was observed for cells that differentiated in AIM and Ult‐G, but the active tension generated by C2C12 cells that differentiated in Ult‐G was only ～25% in the case of myotubes that formed in HS. The addition of Ult‐G to the AIM resulted in improvement of the active tension generation capability, the active tension generated being ～3.4× compared to that in HS. The approach for assessing muscle cell differentiation presented in this study will be suitable for other studies that involve the differentiation of muscle cells. Biotechnol. Bioeng. 2010;107: 894–901. © 2010 Wiley Periodicals, Inc.     

Myoblast adhesion and proliferation on biodegradable polymer films with femtosecond laser‐fabricated micro through‐holes

Controlling cell adhesion and cell differentiation is necessary to fabricate a tissue with arbitrary properties for tissue engineering applications. A substrate with a porous structure as a cell scaffold allows the diffusion of the cell culture medium through the scaffold. In this work, we show that the femtosecond laser fabricated micro through‐holes in biodegradable polymer films, enhance myoblast adhesion, and accelerates proliferation and differentiation. ChR2‐C2C12 and UT‐C2C12 cells were seeded on the films with micro through‐holes each fabricated by a single femtosecond laser pulse. Cell adhesion was enhanced on films with holes fabricated by laser irradiation. In addition, cell proliferation was accelerated on films with micro through‐holes that penetrate the film, compared to on films with micro craters that do not penetrate the film. On films with arrays consisting of micro through‐holes, cells aligned along the arrays and cell fusion was enhanced, indicating the acceleration of cell differentiation.     

Freestanding stacked mesh‐like hydrogel sheets enable the creation of complex macroscale cellular scaffolds

Hydrogel‐based bottom‐up tissue engineering depends on assembly of cell‐laden modules for complex three‐dimensional tissue reconstruction. Though sheet‐like hydrogel modules enable rapid and controllable assembly, they have limitations in generating spatial microenvironments and mass transport. Here, we describe a simple method for forming large‐scale cell‐hydrogel assemblies via stacking cell‐embedded mesh‐like hydrogel sheets to create complex macroscale cellular scaffolds. Freestanding stacked hydrogel sheets were fabricated for long‐term cell culturing applications using a facile stacking process where the micropatterned hydrogel sheets (8.0 mm × 8.7 mm) were aligned using a polydimethylsiloxane drainage well. The stacked hydrogel sheets were precisely aligned so that the openings could facilitate mass transport through the stacked sheets. Despite the relatively large height of the stacked structure (400–700 μm), which is larger than the diffusion limit thickness of 150–200 μm, the freestanding cell‐ydrogel assemblies maintained cell viability and exhibited enhanced cellular function compared with single hydrogel sheets. Furthermore, a three‐dimensional co‐culture system was constructed simply by stacking different cell‐containing hydrogel sheets. These results show that stacked hydrogel sheets have significant potential as a macroscale cell‐culture and assay platform with complex microenvironments for biologically relevant in vitro tissue‐level drug assays and physiological studies.     

Improving survival and efficacy of pluripotent stem cell–derived cardiac grafts

Human embryonic stem cells (hESCs) can be differentiated into structurally and electrically functional myocardial tissue and have the potential to regenerate large regions of infarcted myocardium. One of the key challenges that needs to be addressed towards full‐scale clinical application of hESCs is enhancing survival of the transplanted cells within ischaemic or scarred, avascular host tissue. Shortly after transplantation, most hESCs are lost as a result of multiple mechanical, cellular and host factors, and a large proportion of the remaining cells undergo apoptosis or necrosis shortly thereafter, as a result of loss of adhesion‐related signals, ischaemia, inflammation or immunological rejection. Blocking the apoptotic signalling pathways of the cells, using pro‐survival cocktails, conditioning hESCs prior to transplant, promoting angiogenesis, immunosuppressing the host and using of bioengineered matrices are among the emerging techniques that have been shown to optimize cell survival. This review presents an overview of the current strategies for optimizing cell and host tissue to improve the survival and efficacy of cardiac cells derived from pluripotent stem cells.     

Hybrid chitosan–ß‐glycerol phosphate–gelatin nano‐/micro fibrous scaffolds with suitable mechanical and biological properties for tissue engineering

Scaffold‐based tissue engineering is considered as a promising approach in the regenerative medicine. Graft instability of collagen, by causing poor mechanical properties and rapid degradation, and their hard handling remains major challenges to be addressed. In this research, a composite structured nano‐/microfibrous scaffold, made from a mixture of chitosan–ß‐glycerol phosphate–gelatin (chitosan–GP–gelatin) using a standard electrospinning set‐up was developed. Gelatin–acid acetic and chitosan ß‐glycerol phosphate–HCL solutions were prepared at ratios of 30/70, 50/50, 70/30 (w/w) and their mechanical and biological properties were engineered. Furthermore, the pore structure of the fabricated nanofibrous scaffolds was investigated and predicted using a theoretical model. Higher gelatin concentrations in the polymer blend resulted in significant increase in mean pore size and its distribution. Interaction between the scaffold and the contained cells was also monitored and compared in the test and control groups. Scaffolds with higher chitosan concentrations showed higher rate of cell attachment with better proliferation property, compared with gelatin‐only scaffolds. The fabricated scaffolds, unlike many other natural polymers, also exhibit non‐toxic and biodegradable properties in the grafted tissues. In conclusion, the data clearly showed that the fabricated biomaterial is a biologically compatible scaffold with potential to serve as a proper platform for retaining the cultured cells for further application in cell‐based tissue engineering, especially in wound healing practices. These results suggested the potential of using mesoporous composite chitosan–GP–gelatin fibrous scaffolds for engineering three‐dimensional tissues with different inherent cell characteristics. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 163–175, 2016.     

Alignment of skeletal muscle myoblasts and myotubes using linear micropatterned surfaces ground with abrasives

Alignment of cells plays a significant key role in skeletal muscle tissue engineering because skeletal muscle tissue in vivo has a highly organized structure consisting of long parallel multinucleated myotubes formed through differentiation and fusion of myoblasts. In the present study, we developed an easy, simple, and low‐cost method for aligning skeletal muscle cells by using surfaces with linear microscale features fabricated by grinding. Iron blocks were ground in one direction with three kinds of abrasives (9 µm diamond suspension, #400 sandpaper, and #150 sandpaper) and then used as molds to make micropatterned polydimethylsiloxane (PDMS) substrates (type I, type II, and type III). Observation of the surface topography revealed that the PDMS substrates exhibited different degree of mean roughness (Ra), 0.03 µm for type I, 0.16 µm for type II, and 0.56 µm for type III, respectively. Murine skeletal muscle cell line C2C12 myoblasts were cultured and differentiated on the patterned PDMS substrates, and it was examined whether the alignment of C2C12 myoblasts and myotubes was possible. Although the cell growth and differentiation on the three types of patterned substrates were similar to those on the flat PDMS substrate as a control, the alignment of both C2C12 myoblasts and myotubes was obviously observed on types II and III, but not on type I or the control substrate. These results indicate that surfaces ground with abrasives will be useful for fabricating aligned skeletal muscle tissues. Biotechnol. Bioeng. 2009;103: 631–638. © 2009 Wiley Periodicals, Inc.     

Overexpression of amyloid beta precursor protein enhances expression and secretion of ST6Gal1 in C2C12 myogenic cell line

The Abeta (amyloid‐beta) peptide is derived from the sequential cleavage of AbetaPP (amyloid‐beta precursor protein) by two enzymes, the β‐ and γ‐secretases. The major β‐secretase, identified as the novel transmembrane aspartic protease BACE1 (beta site APP‐cleaving enzyme 1), mediates the primary amyloidogenic cleavage of AbetaPP and initiates the production of Abeta. It has been implicated in the proteolytic processing of another substrate, namely ST6Gal1 (β galactoside α2,6‐sialyltransferase 1), which is the major α2,6‐sialyltransferase responsible for the broad synthesis of glycoproteins and glycolipids. The present study investigated the effect of overexpression of AbetaPP on expression and secretion of ST6Gal1 in skeletal muscle cells by inducing overexpression of wild‐type full‐length 751‐AbetaPP in the mouse myogenic cell line C2C12. Expression and secretion of the ST6Gal1 enzyme were analysed by Western blot and/or immunofluorescence staining. The results of our study demonstrated that AbetaPP overexpression in C2C12 cells increased the expression and the secretion of ST6Gal1 enzyme in vitro.     

Fabrication of a biomimetic spinal cord tissue construct with heterogenous mechanical properties using intrascaffold cell assembly

In tissue engineering studies, scaffolds play a very important role in offering both physical and chemical cues for cell growth and tissue regeneration. However, in some cases, tissue regeneration requires scaffolds with high mechanical properties (e.g., bone and cartilage), while cells need a soft mechanical microenvironment. In this study, to mimic the heterogenous mechanical properties of a spinal cord tissue, a biomimetic rat tissue construct is fabricated. A collagen-coated poly(lactic-co-glycolic acid) scaffold is manufactured using thermally induced phase separation casting. Primary rat neural cells (P01 Wistar rat cortex) with soft hydrogels are later printed within the scaffold using an image-guided intrascaffold cell assembly technique. The scaffolds have unidirectional microporous structure with parallel axial macrochannels (260 ± 4 µm in diameter). Scaffolds showed mechanical properties similar to rat spine (ultimate tensile strength: 0.085 MPa, Young's modulus [stretch]: 0.31 MPa). The bioink composed of gelatin/alginate/fibrinogen is precisely printed into the macrochannels and showed mechanical properties suitable for neural cells (Young's modulus [compressive]: 3.814 kPa). Scaffold interface, cell viability, and immunostaining analyses show uniform distribution of stable, healthy, and elongated neural cells and neurites over 14 culture days in vitro. The results demonstrated that this method can serve as a valuable tool to aid manufacturing of tissue constructs requiring heterogenous mechanical properties for complex cell and/or biomolecule assembly.     

Isolation and culture of the three vascular cell types from a small vein biopsy sample

Summary The availability of small-diameter blood vessels remains a significant problem in vascular reconstruction. In small-diameter blood vessels, synthetic grafts resulted in low patency; the addition of endothelial cells (EC) has clearly improved this parameter, thereby proving the important contribution of the cellular component to the functionality of any construct. Because the optimal source of cells should be autologous, the adaptation of existing methods for the isolation of all the vascular cell types present in a single and small biopsy sample, thus reducing patient’s morbidity, is a first step toward future clinical applications of any newly developed tissue-engineered blood vessel. This study describes such a cell-harvesting procedure from vein biopsy samples of canine and human origin. For this purpose, we combined preexisting mechanical methods for the isolation of the three vascular cell types: EC by scraping of the endothelium using a scalpel blade, vascular smooth muscle cells (VSMC), and perivascular fibroblasts according to the explant method. Once in culture, cells rapidly grew with the high level of enrichment. The morphological, phenotypical, and functional expected criteria were maintained: EC formed cobblestone colonies, expressed the von Willebrand factor, and incorporated acetylated low-density lipoprotein (LDL); VSMC were elongated and contracted when challenged by vasoactive agents; perivascular fibroblasts formed a mechanically resistant structure. Thus, we demonstrated that an appropriate combination of preexisting harvesting methods is suitable to isolate simultaneously the vascular cell types present in a single biopsy sample. Their functional characteristics indicated that they were suitable for the cellularization of synthetic prosthesis or the reconstruction of functional multicellular autologous organs by tissue engineering.     

Analysis of cell growth and diffusion in a scaffold for cartilage tissue engineering

Developments in tissue engineering over the past decade have offered promising future for the repair and reconstruction of damaged tissues. To regenerate three dimensional and weight-bearing implants, advances in biomaterials and manufacturing technologies prompted cell cultivations with natural or artificial scaffolds, in which cells are allowed to proliferate, migrate, and differentiate in vitro. In this article, we develop a mathematical model for cell growth in a porous scaffold. By treating the cell-scaffold construct as a porous medium, a continuum model is set up based on basic principles of mass conservation. In addition to cell growth kinetics, we incorporate cell diffusion in the model to describe the effects of cell random walks. Computational results are compared to experimental data found in the literature. With this model, we are able to investigate cell motility, heterogeneous cell distributions, and non-uniform seeding for tissue engineering applications. Results show that random walks tend to enhance uniform cell spreads in space, which in turn increases the probabilities for cells to acquire nutrients; therefore random walks are likely to be a positive contribution to the overall cell growth on scaffolds.     

Development of a 3D cell culture system for investigating cell interactions with electrospun fibers

There are many variables to be considered in studying how cells interact with 3D scaffolds used in tissue engineering. In this study we investigated the influence of the fiber diameter and interfiber spaces of 3D electrospun fiber scaffolds on the behavior of human dermal fibroblasts. Fibers of two dissimilar model materials, polystyrene and poly-L-lactic acid, with a broad range of diameters were constructed in a specifically developed 3D cell culture system. When fibroblasts were introduced to freestanding fibers, and encouraged to "walk the plank," a minimum fiber diameter of 10 microm was observed for cell adhesion and migration, irrespective of fiber material chemistry. A distance between fibers of up to 200 microm was also observed to be the maximum gap that could be bridged by cell aggregates--a behavior not seen in conventional 2D culture. This approach has identified some basic micro-architectural parameters for electrospun scaffold design and some key differences in fibroblast growth in 3D. We suggest the findings will be of value for optimizing the integration of cells in these scaffolds for skin tissue engineering.     

Bone repair with a form of BMP-2 engineered for incorporation into fibrin cell ingrowth matrices

Most growth factors naturally involved in development and regeneration demonstrate strong binding to the extracellular matrix and are retained there until being locally mobilized by cells. In spite of this feedback between cell activity and growth factor mobilization in the extracellular matrix, this approach has not been extensively explored in therapeutic situations. We present an engineered bone morphogenetic protein-2 (BMP-2) fusion protein that mimics such function in a surgically relevant matrix, fibrin, incorporated into the matrix until it is locally liberated by cell surface-associated proteases. A tripartite fusion protein, denoted TG-pl-BMP-2, was designed and produced recombinantly. An N-terminal transglutaminase substrate (TG) domain provides covalent attachment to fibrin during coagulation under the influence of the blood transglutaminase factor XIIIa. A central plasmin substrate (pl) domain provides a cleavage site for local release of the attached growth factor from the fibrin matrix under the influence of cell-activated plasmin. A C-terminal human BMP-2 domain provides osteogenic activity. TG-pl-BMP-2 in fibrin was evaluated in vivo in critical-size craniotomy defects in rats, where it induced 76% more defect healing with bone at 3 weeks with a dose of 1 mug/defect than wildtype BMP-2 in fibrin. After a dosing study in rabbits, the engineered growth factor in fibrin was evaluated in a prospective clinical study for pancarpal fusion in dogs, where it induced statistically faster and more extensive bone bridging than equivalent treatment with cancellous bone autograft. The strong healing response shown by fibrin including a bound BMP-2 variant suggests that with the combination of bound growth factor and ingrowth matrix, it may be possible to improve upon the natural growth factor and even upon tissue autograft.     

A more cost effective and rapid high percentage germ‐line transmitting chimeric mouse generation procedure via microinjection of 2‐cell, 4‐cell,and 8‐cell embryos with ES and iPS cells

The long‐standing traditional method of delivering embryonic stem (ES) cells adjacent to the inner cell mass (ICM) of blastocysts to generate chimeras improved with the advent of laser‐ or Piezo assisted 8‐cell embryo microinjection. Building on this technology but omitting either the laser or the Piezo to penetrate the zona pellucida and making use of earlier embryonic stages (2‐cell and 4‐cell), we were able to significantly speed up and economize our ES cell microinjection and chimera production throughput. We demonstrate here that embryonic (ES) and induced pluripotent stem (iPS) cells can stay fully pluripotent when delivered into 2‐cell‐ and 4‐cell‐stage embryos, long before they would naturally be incorporated into the ICM of a blastocyst (E3.5) and give rise to high percentage and germline transmitting chimeras. genesis 48:394–399, 2010. © 2010 Wiley‐Liss, Inc.     

Investigating the lysis of small-cell lung cancer cell lines by activated natural killer (NK) cells with a fluorometric assay for NK-cell-mediated cytotoxicity

Activation of natural killer (NK) cells with interleukin-2 (IL-2) and IL-12 leads to an enhanced lysis of tumour cells. We investigated the ability of NK cells, with or without prior activation, to lyse a variety of small-cell lung cancer (SCLC) target cells. Specific lysis was measured with a fluorometric assay for NK-cell-mediated cytotoxicity: target cells were labelled with 3,3′-dioctadecyloxacarbocyanine, a green membrane dye. After co-incubation with NK cells, dead target cells were stained with propidium iodide, a red DNA dye that only penetrates dead cells. Of all eight SCLC cell lines tested, three were susceptible to lysis by non-activated NK cells, three were only susceptible to lysis by NK cells activated with IL-2 and IL-12 and two were not even susceptible to lysis by activated NK cells. The differences in target cell susceptibility showed no correlation with the expression of MHC-I on the surface of the target cells or with the expression of the adhesion molecules CD50, CD54, CD58 or CD102. Comparing the kinetics of the lysis of one SCLC cell line sensitive to non-activated NK cells and one sensitive only to activated NK cells, we found that maximum lysis of the former was obtained after 1 h, whereas significant lysis of the latter was only obtained after 4 h of incubation. This might be due to different mechanisms engaged in target cell lysis. Received: 23 December 1998 / Accepted: 8 April 1999     

Design and validation of a pulsatile perfusion bioreactor for 3D high cell density cultures

This study presents the design and validation of a pulsatile flow perfusion bioreactor able to provide a suitable environment for 3D high cell density cultures for tissue engineering applications. Our bioreactor system is mobile, does not require the use of traditional cell culture incubators and is easy to sterilize. It provides real‐time monitoring and stable control of pH, dissolved oxygen concentration, temperature, pressure, pulsation frequency, and flow rate. In this bioreactor system, cells are cultured in a gel within a chamber perfused by a culture medium fed by hollow fibers. Human umbilical vein endothelial cells (HUVEC) suspended in fibrin were found to be living, making connections and proliferating up to five to six times their initial seeding number after a 48‐h culture period. Cells were uniformly dispersed within the 14.40 mm × 17.46 mm × 6.35 mm chamber. Cells suspended in 6.35‐mm thick gels and cultured in a traditional CO₂ incubator were found to be round and dead. In control experiments carried out in a traditional cell culture incubator, the scarcely found living cells were mostly on top of the gels, while cells cultured under perfusion bioreactor conditions were found to be alive and uniformly distributed across the gel. Biotechnol. Bioeng. 2009; 104: 1215–1223. © 2009 Wiley Periodicals, Inc.     

High‐throughput screening of a small molecule library for promoters and inhibitors of mesenchymal stem cell osteogenic differentiation

The use of high‐throughput screening (HTS) techniques has long been employed by the pharmaceutical industry to increase discovery rates for new drugs that could be useful for disease treatment, yet this technology has only been minimally applied in other applications such as in tissue regeneration. In this work, an assay for the osteogenic differentiation of human mesenchymal stem cells (hMSCs) was developed and used to screen a library of small molecules for their potential as either promoters or inhibitors of osteogenesis, based on levels of alkaline phosphatase activity and cellular viability. From a library of 1,040 molecules, 36 promoters, and 20 inhibitors were identified as hits based on statistical criteria. Osteopromoters from this library were further investigated using standard culture techniques and a wider range of outcomes to verify that these compounds drive cellular differentiation. Several hits led to some improvement in the expression of alkaline phosphatase, osteogenic gene expression, and matrix mineralization by hMSCs when compared to the standard dexamethasone supplemented media and one molecule was investigated in combination with a recently identified biodegradable and osteoconductive polymer. This work illustrates the ability of HTS to more rapidly identify potential molecules to control stem cell differentiation. Biotechnol. Bioeng. 2011; 108:163–174. © 2010 Wiley Periodicals, Inc.